BACKGROUND: Women’s sexual wellbeing is an important determinant of overall health and quality of life across the life course. Yet the factors associated with women’s levels of sexual activity and sexual function in midlife are little understood. OBJECTIVE: This study sought to assess the prevalence of recent sexual activity and sexual dysfunction symptoms among middle-aged women and evaluate the associations of partner status, menopause, and health status factors with sexual dysfunction. METHODS: Participants of this cross-sectional study were 68,131 women who responded to the 2013 Nurses’ Health Study II observational cohort questionnaire when they were age 48–68 years. Sexual activity and dysfunction symptoms were assessed with the Female Sexual Function Index (FSFI-6). Age adjusted multivariable regression models estimated risk ratios (RRs) for the association of health-related factors with past-month sexual dysfunction symptoms among women who were sexually active over the past month, overall and stratified by partner status. RESULTS: 73% of middle-aged women were sexually active (N=49,701) and 50% of sexually active women reported symptoms of sexual dysfunction. Symptoms of sexual dysfunction were less common among unpartnered than partnered women (42% vs. 51%, p